This invention is in the field of plant molecular biology. More specifically, this invention pertains to nucleic acid fragments encoding 1-deoxy-D-xylulose 5-phosphate synthase in plants and seeds.
Isoprenoids comprise the largest family of natural products, including numerous secondary compounds, which play different functional roles in plants such as hormones, photosynthetic pigments, electron carriers, and structural components of membranes. The fundamental unit in isoprenoid biosynthesis, isopentenyl diphosphate (IPP), is normally synthesized by the condensation of acetyl CoA through the mevalonate pathway. In many organisms including several bacteria, algae and plant plastids, IPP is synthesized by a mevalonate-independent pathway. The initial step, in this pathway is the condensation of pyruvate and glyceraldehyde 3-phosphate to form 1-deoxy-D-xylulose 4-phosphate which behaves as the precursor for IPP, thiamine (vitamin B1), or pyridoxine (vitamin B2). This initial step is catalyzed by 1-deoxy-D-xylulose 5-phosphate synthase (DXPS), a member of a distinct proteins family. In E. coli DXPS shows sequence similarity to both transketolases and the E1 subunit of pyruvate dehydrogenase (Sprenger (1997) Proc. Natl. Acad. Sci. USA 94:12857-12862).
The present invention relates to isolated polynucleotides comprising a nucleotide sequence encoding a first polypeptide of at least 170 amino acids that has at least 95% identity based on the Clustal method of alignment when compared to a polypeptide selected from the group consisting of a corn 1-deoxy-D-xylulose 5-phosphate synthase polypeptide of SEQ ID NOs:2, 4, 18, 20, 22, and 24, a rice 1-deoxy-D-xylulose 5-phosphate synthase polypeptide of SEQ ID NOs:6, 8, 26, and 28, a soybean 1-deoxy-D-xylulose 5-phosphate synthase polypeptide of SEQ ID NOs:10 and 12, a wheat 1-deoxy-D-xylulose 5-phosphate synthase polypeptide of SEQ ID NO:14, 16, 30, and 32. The present invention also relates to an isolated polynucleotide comprising the complement of the nucleotide sequences described above.
It is preferred that the isolated polynucleotide of the claimed invention consists of a nucleic acid sequence selected from the group consisting of SEQ ID NOs:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, and 31 that codes for the polypeptide selected from the group consisting of SEQ ID NOs:2; 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, and 32. The present invention also relates to an isolated polynucleotide comprising a nucleotide sequences of at least one of 40 (preferably at least one of 30) contiguous nucleotides derived from a nucleotide sequence selected from the group consisting of SEQ ID NOs:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, and the complement of such nucleotide sequences.
The present invention relates to a chimeric gene comprising an isolated polynucleotide of the present invention operably linked to suitable regulatory sequences.
The present invention relates to an isolated host cell comprising a chimeric gene of the present invention or an isolated polynucleotide of the present invention. The host cell may be eukaryotic, such as a yeast or a plant cell, or prokaryotic, such as a bacterial cell. The present invention also relates to a virus, preferably a baculovirus, comprising an isolated polynucleotide of the present invention or a chimeric gene of the present invention.
The present invention relates to a process for producing an isolated host cell comprising a chimeric gene of the present invention or an isolated polynucleotide of the present invention, the process comprising either transforming or transfecting an isolated compatible host cell with a chimeric gene or isolated polynucleotide of the present invention.
The present invention relates to a 1-deoxy-D-xylulose 5-phosphate synthase polypeptide of at least 170 amino acids comprising at least 95% homology based on the Clustal method of alignment compared to a polypeptide selected from the group consisting of SEQ ID NOs:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, and 32.
The present invention relates to a method of selecting an isolated polynucleotide that affects the level of expression of a 1-deoxy-D-xylulose 5-phosphate synthase polypeptide in a host cell, the method comprising the steps of: (a) constructing an isolated polynucleotide of the present invention or an isolated chimeric gene of the present invention; (b) introducing the isolated polynucleotide or the isolated chimeric gene into a host cell; (c) measuring the level a 1-deoxy-D-xylulose 5-phosphate synthase polypeptide in the host cell containing the isolated polynucleotide; and (d) comparing the level of a 1-deoxy-D-xylulose 5-phosphate synthase polypeptide in the host cell containing the isolated polynucleotide with the level of a 1-deoxy-D-xylulose 5-phosphate synthase polypeptide in a.host cell that does not contain the isolated polynucleotide.
The present invention relates to a method of obtaining a nucleic acid fragment encoding a substantial portion of a 1-deoxy-D-xylulose 5-phosphate synthase polypeptide gene, preferably a plant 1-deoxy-D-xylulose 5-phosphate synthase polypeptide gene, comprising the steps of: synthesizing an oligonucleotide primer comprising a nucleotide sequence of at least one of 40 (preferably at least one of 30) contiguous nucleotides derived from a nucleotide sequence selected from the group consisting of SEQ ID NOs:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, and the complement of such nucleotide sequences; and amplifying a nucleic acid fragment (preferably a cDNA inserted in a cloning vector) using the oligonucleotide primer. The amplified nucleic acid fragment preferably will encode a portion of a 1-deoxy-D-xylulose 5-phosphate synthase amino acid sequence.
The present invention also relates to a method of obtaining a nucleic acid fragment encoding all or a substantial portion of the amino acid sequence encoding a 1-deoxy-D-xylulose 5-phosphate synthase polypeptide comprising the steps of: probing a cDNA or genomic library with an isolated polynucleotide of the present invention; identifying a DNA clone that hybridizes with an isolated polynucleotide of the present invention; isolating the identified DNA clone; and sequencing the cDNA or genomic fragment that comprises the isolated DNA clone.
A further embodiment of the instant invention is a method for evaluating at least one compound for its ability to inhibit the activity of a 1-deoxy-D-xylulose 5-phosphate synthase, the method comprising the steps of: (a) transforming a host cell with a chimeric gene comprising a nucleic acid fragment encoding a 1-deoxy-D-xylulose 5-phosphate synthase, operably linked to suitable regulatory sequences; (b) growing the transformed host cell under conditions that are suitable for expression of the chimeric gene wherein expression of the chimeric.gene results in production of 1-deoxy-D-xylulose 5-phosphate synthase in the transformed host cell; (c) optionally purifying the 1-deoxy-D-xylulose 5-phosphate synthase expressed by the transformed host cell; (d) treating the 1-deoxy-D-xylulose 5-phosphate synthase with a compound to be tested; and (e) comparing the activity of the 1-deoxy-D-xylulose 5-phosphate synthase that has been treated with a test compound to the activity of an untreated 1-deoxy-D-xylulose 5-phosphate synthase, thereby selecting compounds with potential for inhibitory activity.